Hydrolysis of pyrethroids by carboxylesterases from Lucilia cuprina and Drosophila melanogaster with active sites modified by in vitro mutagenesis
The cloned genes encoding carboxylesterase E3 in the blowfly Lucilia cuprina and its orthologue in Drosophila melanogaster were expressed in Sf9 cells transfected with recombinant baculovirus. Resistance of L. cuprina to organophosphorus insecticides is due to mutations in the E3 gene that enhance t...
Saved in:
Published in | Insect biochemistry and molecular biology Vol. 35; no. 6; pp. 597 - 609 |
---|---|
Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Elsevier Ltd
01.06.2005
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | The cloned genes encoding carboxylesterase E3 in the blowfly
Lucilia cuprina and its orthologue in
Drosophila melanogaster were expressed in Sf9 cells transfected with recombinant baculovirus. Resistance of
L. cuprina to organophosphorus insecticides is due to mutations in the E3 gene that enhance the enzyme's ability to hydrolyse insecticides. Previous in vitro mutagenesis and expression of these modifications (G137D, in the oxyanion hole and W251L, in the acyl pocket) have confirmed their functional significance. We have systematically substituted these and nearby amino acids by others expected to affect the hydrolysis of pyrethroid insecticides. Most mutations of G137 markedly decreased pyrethroid hydrolysis. W251L was the most effective of five substitutions at this position. It increased activity with
trans permethrin 10-fold, and the more insecticidal
cis permethrin >130-fold, thereby decreasing the
trans:
cis hydrolysis ratio to only 2, compared with >25 in the wild-type enzyme. Other mutations near the bottom of the catalytic cleft generally enhanced pyrethroid hydrolysis, the most effective being F309L, also in the presumptive acyl binding pocket, which enhanced
trans permethrin hydrolysis even more than W251L. In these assays with racemic 1RS
cis and 1RS
trans permethrin, two phases were apparent, one being much faster suggesting preferential hydrolysis of one enantiomer in each pair as found previously with other esterases. Complementary assays with individual enantiomers of deltamethrin and the dibromo analogue of
cis permethrin showed that the wild type and most mutants showed a marked preference for the least insecticidal 1S configuration, but this was reversed by the F309L substitution. The W251L/F309L double mutant was best overall in hydrolysing the most insecticidal 1R
cis isomers. The results are discussed in relation to likely steric effects on enzyme–substrate interactions, cross-resistance between pyrethroids and malathion, and the potential for bioremediation of pyrethroid residues. |
---|---|
Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0965-1748 1879-0240 |
DOI: | 10.1016/j.ibmb.2005.02.018 |